SEM imaging 101: The Secrets to Scanning Electron Microscopy
- Sophie O'Brien
- May 19
- 4 min read
When we think of microscopes, we often picture the run-of-the-mill compound microscopes that live in your high school chemistry teacher’s lab. While perfectly capable of analyzing 2D plant cells in biology class, traditional light microscopes can only go so far in the world of cutting-edge research and nanotechnology. In order to analyze samples at the nanometer scale—far beyond the capabilities of what the naked eye or visible light can see—Scanning Electron Microscopy (SEM) is essential.
What are SEMs?
SEMs are microscopes that do not rely on visible light but rather use a focused beam of electrons to create images of a specimen’s topographical surface. Electrons have a wavelength that is orders of magnitude smaller than visible light, allowing SEMs to produce high-quality 3D images at extremely high magnifications from 10x to 1,000,000x, depending on the instrument. The first SEM prototype was actually invented back in 1937 by German physicist Manfred von Ardenne; however, it took several decades for the technology to evolve into the widely adopted, commercially available machines we use today.
It is important to distinguish between the different kinds of SEM machines and their respective functions. The two main types are the conventional SEM and the Environmental SEM (ESEM). The conventional SEM can only analyze samples that are solid, dry, and electrically conductive (non-conductive materials can be coated with gold or carbon before use). The ESEM, however, allows for biological and wet samples that are not electrically conductive and can introduce water into the gas chamber, making the ESEM valuable for biological science applications to observe living cells or hydrated materials. It is important to identify which type of SEM machine you are using in your lab for the sake of the experiment, safety, and well-being of the machine, as the settings and guidelines for using each would be different. This blog post and the subsequent parts will mainly focus on the conventional SEM.
How do SEMs work?
The first step of using an SEM machine actually begins outside the equipment room. Before imaging, samples need to be coated with a thin layer of a conductive material, such as gold or carbon, to prevent the buildup of electrical charge that could damage the specimen and to facilitate interactions with the electron beam and surface for signal quality. Note that this step can be skipped when using Environmental SEMs (ESEMs) or certain advanced lenses like Variable Pressure (VP) modes.
Another crucial background step to know is that SEMs must function in a vacuum-sealed environment (this occurs during setup when you vent and pump the SEM machine). The vacuum seal ensures that nothing interferes with the electron pathway, allowing for the collection of clear, interpretable signals.
An SEM begins by generating a beam of electrons with an electron gun that is funneled through an aperture for precision to hit the sample. This highly focused electron beam uses a raster scan pattern to map the entire surface area of interest and create an image (think of sweeping a flashlight in a dark cave to illuminate its contents). When the beam strikes the sample, it generates different signals and sample-electron interactions, namely secondary electrons, backscattered electrons, and characteristic X-rays. These interactions are picked up by their respective detector.
The three primary types of signals and their associated detectors include:
Secondary Electron Detector: Captures low-energy electrons emitted from the sample surface for surface topography. This is the most commonly used detector in SEM.
Backscattered Electron Detector: Detects electrons that are reflected off and back from the sample.
Energy Dispersive X-ray Spectroscopy (EDS): Measures characteristic X-rays emitted when electrons dislodge inner-shell electrons in atoms, providing the composition of elements based on atomic number. This scanner is used typically in tandem with the other two detectors for comprehensive analysis.
When and in what fields are SEMs used?
SEM machines are an interdisciplinary device that technically any scientist interested in analyzing a material can use —the only qualifications for handling a machine are proper training and curiosity! However, there are some (not all!) fields of research where SEM imaging is more commonly used:
Materials science: Studying material properties, including cracks, surface fractures, and how new materials fit together.
Bioinspired materials science: SEMs allow scientists to discover and visualize the structures and matrixes of novel nanocomposite materials created to mimic biological structures.
Semiconductor research: Inspecting surfaces and properties on electrical devices and on microchips)
Geology/Planetary sciences: Studying rare minerals or unknown samples from space
Forensics: Analyzing crime scene evidence, like blood residue or hair.
*Note in applications like forensics or samples from space, where it is imperative not to alter the original sample with conductive coating, variable pressure modes would most likely be utilized.
Sophie’s SEM imaging tips and tricks!
While I’m not an expert just yet, here are some helpful SEM tips that I’ve learned from my amazing supervisors and from spending my own time with the machine.
Make sure the specimen is properly prepared beforehand: 1) Solid (no waxes/oils/liquids), 2) Dry (place for about 24 hours in a 60℃ oven), 3) Conductive (with gold/carbon).
It is good practice to focus at twice the magnification you want to capture at and then zoom out. For example, to take a picture at 100kX, play with the focus settings at 200kX by finding a small interesting feature on the specimen, and then go back out.
Take a water/snack break (outside of the lab, of course…). It can get tiring sitting in front of the SEM screen for hours. Don’t be afraid to take a quick break before getting back to work! :)
Here are some other helpful resources for understanding SEM machines:
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